Fluorogenic kinetic assay for high-throughput discovery of stereoselective ketoreductases relevant to pharmaceutical synthesis

Enantiomerically pure 1-(6-methoxynaphth-2-yl) and 1-(6-(dimethylamino)naphth-2-yl) carbinols are fluorogenic substrates for aldo/keto reductase (KRED) enzymes, which allow the highly sensitive and reliable determination of activity and kinetic constants of known and unknown enzymes, as well as an immediate enantioselectivity typing. Because of its simplicity in microtiter plate format, the assay qualifies for the discovery of novel KREDs of yet unknown specificity among this vast enzyme superfamily. The suitability of this approach for enzyme typing is illustrated by an exemplary screening of a large collection of short-chain dehydrogenase/reductase (SDR) enzymes arrayed from a metagenomic approach. We believe that this assay format should match well the pharmaceutical industry’s demand for acetophenone-type substrates and the continuing interest in new enzymes with broad substrate promiscuity for the synthesis of chiral, non-racemic carbinols

Increase of enzyme activity through specific covalent modification with fragments

Modulation of enzyme activity is a powerful means of probing cellular function and can be exploited for diverse applications. Here, we explore a method of enzyme activation where covalent tethering of a small molecule to an enzyme can increase catalytic activity (k cat/K M) up to 35-fold. Using a bacterial glycoside hydrolase, BtGH84, we demonstrate how small molecule "fragments", identified as activators in free solution, can be covalently tethered to the protein using Michael-addition chemistry. We show how tethering generates a constitutively-activated enzyme-fragment conjugate, which displays both improved catalytic efficiency and increased susceptibility to certain inhibitor classes. Structure guided modifications of the tethered fragment demonstrate how specific interactions between the fragment and the enzyme influence the extent of activation. This work suggests that a similar approach may be used to modulate the activity of enzymes such as to improve catalytic efficiency or increase inhibitor susceptibility

Minimalist Protein Engineering of an Aldolase Provokes Unprecedented Substrate Promiscuity

Application of aldolases for the asymmetric synthesis of multifunctional chiral products is hampered by their reputed strict nucleophile (=aldol donor) specificity owing to a mechanistic requirement for creating a carbanion nucleophile in aqueous medium. Here we report that a minimalist engineering can extensively broaden the substrate scope of native d-fructose-6-phosphate aldolase (FSA) from Escherichia coli, for which hydroxyacetone is the most proficient substrate, to accept an unprecedented wide variety of alternative nucleophiles. By single- or double-space-generating mutations using simple conservative Leu to Ala replacement of active site residues, we found enzyme variants to efficiently convert larger ketols and bioisosteric ether components with up to seven skeletal atoms, including linear and branched-chain structures. All reactions occurred with full retention of the natural d-threo diastereospecificity. These FSA variants open new avenues toward the synthesis of novel product families that hitherto were inaccessible by biological catalysis.This work was funded by the Bundesministerium für Bildung und Forschung (BMBF grant 0315775B PT-J to W.-D.F.) and the Ministerio de Economı́a y Competitividad (MINECO) (grant CTQ2012-31605 to P.C.), within the transnational Eurotrans-Bio framework, as well as by student exchange funds from the DAAD (grant PPP-50749958 to W.-D.F.), Acciones Integradas (MINECO; grant AIB2010DE-00405 to P.C.), and COST action CM1303 Systems Biocatalysis.Peer reviewe

Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

A protein engineering approach for expanding the substrate scope of the (S)-selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl- and benzyl-substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2-diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)-1,2-diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20-30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzyl-substituted primary amines

Reactor and microreactor performance and kinetics of the aldol addition of dihydroxyacetone to benzyloxycarbonyl-N-3-aminopropanal catalyzed by D-fructose-6-phosphate aldolase variant A129G

D-Fagomine is an iminosugar found in nature that lowers blood glucose peak after meal and reduces fat-induced weight gain and insulin resistance. The immediate precursor of D-fagomine is accessible straightforward via D-fructose-6-phosphate aldolase catalyzed aldol addition of dihydroxyacetone to Cbz-N-3-aminopropanal (Cbz = benzyloxycarbonyl). In this work, the performance and kinetics of the abovementioned reaction catalyzed by FSA A129G variant was studied. The reaction was investigated in two reactors; batch reactor and microreactor, and kinetic parameters were estimated from the steady state experiments in the batch reactor. This enzyme has improved stability, better K m value for Cbz-N-3-aminopropanal and lower retro-aldol activity in comparison with previously studied aldolase variants FSA A129S and A129S/A165G. Mathematical model for both reactors was developed and validated experimentally. © 2018, © 2018 Taylor & Francis Group, LLC.The Croatian Ministry of Science Education and Sports and Spanish National Research Council financed this work in the Frame of the ERA-IB project: EIB. 10.012.MicroTechEnz. The support of the Ministerio de Economía y Competitividad (MINECO), the Fondo Europeo de Desarrollo Regional (FEDER) (grant no. CTQ2015-63563-R, and ERA-IB MICINN PIM2010EEI-00607) is acknowledged. Authors thank COST Action CM0701 and Susana Amézqueta for the assessment of purity of the Bioglane S.L.N.E. materials used in this work.Peer reviewe

Efficient one-pot tandem biocatalytic process for a valuable phosphorylated C8 d-ketose: d-glycero-d-altro-2-octulose 8-phosphate.

International audienc

Efficient biocatalytic processes for highly valuable terminally phosphorylated C5 to C9 D-ketoses.

International audienceA green enzymatic strategy for the synthesis of terminally phosphorylated C5 to C9 naturally occurring D-ketose phosphates and analogues was developed using D-fructose-6-phosphate aldolase (FSA) as a catalyst. This enzyme has stereoselectively catalysed aldol reactions between glycolaldehyde phosphate or ribose-5-phosphate as an acceptor substrate and dihydroxyacetone, hydroxyacetone or hydroxybutanone as a donor. Furthermore, D-glycero-D-altro-2-octulose 8-phosphate was obtained using a straightforward one-pot domino biocatalytic system involving FSA, ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase controlling five contiguous asymmetric centres and starting from achiral material

Engineering the donor selectivity of D-Fructose-6-Phosphate Aldolase for Biocatalytic Asymmetric Cross-Aldol additions of glycolaldehyde.

International audienceD-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80–98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity